Pure vitamin b12 and method of making same



United States Pate PURE VITAMIN B AND METHOD OF MAKING SAME GerardNomine, Noisy-le-Sec, Lucien Penasse, Paris, and

Pierre Barthelemy, St.-Germain-en-Laye, France, assignors to UCLAF,Paris, France, a body corporate 'of France No Drawing. Filed Feb. 20,1958, Ser. No. 716,271 Claims priority, application France Mar. 13, 19576 Claims. (Cl. 260211.5)

This invention relates to pure vitamin B -and to a method of makingsame. 7

In our copending application Serial No. 602,874, filed August 8, 1956,and of which the present is a continuaa MNMOM CM GHG )imam.

The vitamin B may be regenerated from these complexes byredissolution'in water and the addition of a water-soluble solventwherein it is insoluble.

It is an object of the present invention to provide a I method ofobtaining pure vitamin B using polyphenols in the method to obtainvitamin B phenol complexes.

One other object of the present invention is to provide hydrated vitaminB phenolic complexes as new products of manufacture.

Other objects and features will become apparent from 7 l the followingdescription of the invention and the accompanying claims.

It has been found in accordance with the present invention that otherphenols, notably dior triphenols, namely orcinol, phloroglucinol orhydroquinone, designated by the letter R in the formula below, are alsocapable of supplying crystallized complexes of the formula:

wherein n represents the numeral 2 or 4 and x represents a value rangingbetween 4 and 11. These compounds may be used to isolate vitamin B fromthe aqueous concentrates of the latter.

The invention herein, therefore, also relates to the hydrated phenoliccomplexes prepared according to the process of the invention as newcommercial products. In order to prepare these hydrated phenoliccomplexes, a desired quantity of phenol is added to an aqueous solutionof vitamin B said quantity being below the limit of solubility of thephenol in question in water. The complex that forms is separated(centrifuged). By being suspended in water, it separates (dissociates),vitamin B is released and dissolves. By adding a water-soluble solventwherein vitamin B is insoluble, the latter precipitates.

In accordance with the above description there are given the followingexamples which serve to illustrate the invention without howeverlimiting its scope. In particular, it is possible to change the originand the concentration of the vitamin B solution, the concentration ofphenol,

- -Pa tented Mar. 21, 1961 the quantities of water used to redissolvethe hydrated phenolic complex of vitamin B and the nature of the solventused for reprecipitating vitamin B or reverse the order of introducingthe compounds without thereby exceeding'or departing from the scope ofthe invention.

. EXAMPLE 1 Formation of the hydrated vitamin B -orcinol complex 800trig. orcinol are added to 20 cc. of an aqueous solution of 200 mg.cyanocobalamine, and the mixture is made lukewarm until it is dissolved.The mixture is left in a refrigerator overnight, then is separated orcentrifuged anddried in vacuo at room temperature. crystals thusobtained are washed with ether in order 'to The resultant eliminatepossible'excess orcinol and are again dried in .vacuo at roomtemperature. The 'dark red crystals thus formed, about 271 mg., areinsoluble in acetone and ether, but soluble in alcohol. They separate inwater releasing vitamin B 'The crystals comprise 70.1% of vitamin B Whendried in vacuo at 60 C., they lose 9.9% of their weight in water. Theproduct dried in this manner comprises 76% of vitamin B which permitsone to conclude that this is a combination of 4 molecules of orcinolandone molecule of vitamin B An analysis shows vitamin B 4 orcinol in theanhy- .drous product.

As calculated: C=59%; H=6.53%; N=10.58%; B

As found: C=58.8%; H=6.6%; N=l1.l%; B =76%.

Hydration after loss: 1 11-1 0.

This compound has not been described in the past and is new. Upondissolution in 100 parts of water and on addition of dioxane or acetone,10 volumes, there is recovered crystallized vitamin B 'titrating above95% and with a practically quantitative yield.

EXAMPLE 2 Formation of the hydrated vitamin B -phloroglucinol complexThe method of Example 1 is followed herein and 200 mg. .ofphloroglucinol are used for 2O cc. of an aqueous An analysis of theanhydrous product vitamin B -2 phloroglucinol shows:

As calculated: C=56.03%; H=6.27%; N=12.l9%;

As found: C=56%; H=6.2%; N=11.9%; B

This compound has not been described in the past and is new.

EXAMPLE 3 Formation of the hydrated vitamin B -hydroquinone complex Themethod of Example 1 is followed herein, and 1 g. of hydroquinone is usedfor 20 cc. of an aqueous solution of 200 mg. vitamin B There are therebyproduced 142 mg. of dark red crystals insoluble in acetone and ether,but soluble in alcohol, and which separate in water releasing vitamin BThey comprise 72.5% vitamin B When dried in vacuo at 60 C. they lose7.6% of their weight in water. The dehydrated product comprises 78%vitamin B which permits one to conclude that this is a combination of 4'molecules. of hydroqui'none and one molecule of vitamin B On analysisthere is obtained vitamin B --4 hydroquinone which shows:

As calculated: C=58.19%; H=6.28%; N: 10.92%; B

As found: C=58%; H=6.1%; N=11.2%; B =78%.

This product has not been described in the past and is new.

The vitamin B formula indicated in the copending patent applicationSerial No. 602,874 is c H N O PCo. Since this formula has been modifiedby Crowfoot Hodgkin and coll. (Nature, 1956, 178,. 65.), who ascribe tovitamin B the. formula CG3HBBN14014PCO, the analytical figures have beencalculated on the basis of this new formula.

While several examples of the invention have been described, it is to benoted that they are not restrictive and that changes as to form and useof materials may be made without departing from the spirit and scope ofthe invention as claimed.

We claim:

1. The method of preparing substantially pure cyanocobalamine complexwhich comprises reacting an aqueous solution of vitamin B containing aminimum of 0.05 of vitamin B with an amount of a phenol selected fromthe group consisting of orcinol, phloroglucinol and hydroquinone, whichis below the limit of solubility of the phenol in an aqueous solution ofvitamin B crystallizing the resultant vitamin B phenol complex,separating, redissolving in water, precipitating the cyanocobalaminetherein by the addition of a water miscible solvent selected from thegroup consisting of dioxane and acetone.

2. In a process of preparing substantially pure cyanocobalamine thesteps which comprise reacting an aqueous solution of vitamin Bcontaining at least 0.05% ofvitamin B with an amount of orcinol which isbelow the limit of its solubility in an aqueous solution of vitamin Bcrystallizing the resulting hydrated vitamin B orcinol complex compound,separating said crystals, redissolving the same in water andprecipitating the cyanocobalamine.

3. In a process of preparing substantially pure cyanocobalamine thesteps which comprise reacting an aqueous solution of vitamin Bcontaining at least 0.05% of vitamin B with an amount of phloroglucinolwhich is below the limit of its solubility in an aqueous solution ofvitamin B crystallizing the resulting hydrated vitamin B phloroglucinolcomplex compound, separating said crystals, redissolving the same inwater, and precipitating the cyanocobalamine.

4. In a process of preparing substantially pure cyanocobalamine, thesteps which comprise reactingan aqueous solution of vitamin B containingat: least.0.05% of vitamin B with an amount of hydroquinone which isbelow the limit of its solubility in an aqueous solution of vitamin B12,crystallizing the resulting hydrated vitamin B hydroquinone complexcompound, separating said crystals, redissolving the same in water, andprecipitating the cyanocobalamine.

5. In a process of preparing substantially pure cyanobalamine, the stepswhich comprise reacting an aqueous solution of vitamin B containing atleast 0.05% of vitamin B With a polyphenol selected from the group consisting of a phenol having two phenolic hydroxyl groups and of a phenolhaving three phenolic hydroxyl groups, said polyphenol being added inan. amount which is below the limit of its solubility in an aqueoussolution of vitamin B crystallizing the resulting hydrated vitamin Bphenolic complex compound, separating said crystals, redissolvingthesamein water, and' precipitating the cyanocobalamine.

6. A hydrated crystalline vitamin B -phenolic complex compoundcorresponding to the formula n is an integer selected from the integers2 and 4, and x is an integer ranging from 4-11.

References Cited in the file of this patent UNITED STATES PATENTSBernhauer et a1 Feb. 11, 1958 OTHER REFERENCES Bernhauer et al.:Angewandte Chemie, 66 (24), 776- 780 (1954).

1. THE METHOD OF PREPARING SUBSTANTIALLY PURE CYANOCOBALAMINE COMPLEXWHICH COMPRISES REACTING AN AQUEOUS SOLUTION OF VITAMIN B12 CONTAINING AMINIMUM OF 0.05% OF VITAMIN B12 WITH AN AMOUNT OF A PHENOL SELECTED FROMTHE GROUP CONSISTING OF ORCINOL, PHLOROGLUCINOL AND HYDROQUINONE, WHICHIS BELOW THE LIMIT OF SOLUBILITY OF THE PHENOL IN AN AQUEOUS SOLUTION OFVITAMIN B12, CRYSTALLIZING THE RESULTANT VITAMIN B12 PHENOL COMPLEX,SEPARATING, REDISSOLVING IN WATER, PRECIPITATING THE CYANOCOBALAMINETHEREIN BY THE ADDITION OF A WATER MISCIBLE SOLVENT SELECTED FROM THEGROUP CONSISTING OF DIOXANE AND ACETONE.